The emergence of antibiotic resistant bacteria is one of the greatest threats to human health in the 21st century. Among these bacteria, vancomycin-resistant enterococci (VRE) are one of the most challenging organisms in clinical settings. Indeed, vancomycin-resistant Enterococcus faecium have been designated by the Infectious Disease Society of America as one of the superbugs against which new therapies are urgently needed. Currently, one of the antibiotics with in vitro bactericidal against VRE is daptomycin (DAP), a lipopeptide antibiotic that has become the key front-line antimicrobial against these organisms due to the paucity of other options. However, the main challenge when using DAP against VRE is the development of resistance during therapy. We have identified a cluster of genes in enterococci (designated liaFSR), encoding a three- component regulatory system likely involved in the cell envelope bacterial response to antibiotics which is highly conserved in Gram-positive pathogens. Our preliminary data indicate that LiaR (encoding the response regulator of the system) is the master regulator of antibiotic resistance in DAP-resistant VRE since deletion of the gene reversed resistance to DAP in several strains of E. faecalis and E. faecium, independent of the genetic background. Therefore, we hypothesize that inhibition of the LiaR response would disarm VRE by altering the bacterial adaptive response to the attack by antimicrobials. In this proposal we seek to develop a new class of molecules (designated anti-adaptation antibiotics) by developing molecules that inhibit LiaR. These compounds are expected to act synergistically with other antibiotics (e.g., DAP) and prevent emergence of resistance. We will develop our experimental approach in two phases. In the R21 phase, the Specific Aims include, i) characterization of the role of LiaR in vancomycin-resistant E. faecium by generating additional liaR gene knockouts from E. faecium, select in vitro DAP-resistant mutants in a liaR deletion mutant, and investigate the liaR regulon as well as developing biochemical tests to evaluate its activity and obtain structural data as the basis for structure-activity relationship studies, and ii) develop a high throughput strategy to identify compounds that are likely to inhibit LiaR activity using reversion of DAP susceptibility as a surrogate marke. The screening will be performed using compound libraries present at research sites associated with this application. Compounds identified would be tested for their ability to interact with LiaR using biochemical and structural data developed in Sp Aim (i). In the R33 phase, we will seek to i) optimize the chemical development of LiaR inhibitors by improving the properties of the hit compounds based on the biochemical and structural information generated in the R21 phase, and ii) assess the in vivo toxicity and efficacy of novel compounds by performing proof of principle in vivo studies using a mouse peritonitis model with calculation of the maximal tolerated dose in animals. At the end of this proposal, we expect to deliver novel compounds that could be used as anti-adaptation antibiotics for further clinical development.